HTRA1 promoter polymorphism predisposes Japanese to age-related macular degeneration.

Purpose To study the effect of candidate single nucleotide polymorphisms (SNPs) on chromosome 10q26, recently shown to be associated with wet age-related macular degeneration (AMD) in Chinese and Caucasian cohorts, in a Japanese cohort. Methods Using genomic DNA isolated from peripheral blood of wet AMD cases and age-matched controls, we genotyped two SNPs, rs10490924, and rs11200638, on chromosome 10q26, 6.6 kb and 512 bp upstream of the HTRA1 gene, respectively, using temperature gradient capillary electrophoresis (TGCE) and direct sequencing. Association tests were performed for individual SNPs and jointly with SNP complement factor H (CFH) Y402H. Results The two SNPs, rs10490924 and rs11200638, are in complete linkage disequilibrium (D'=1). Previous sequence comparisons among seventeen species revealed that the genomic region containing rs11200638 was highly conserved while the region surrounding rs10490924 was not. The allelic association test for rs11200638 yielded a p-value <10-11. SNP rs11200638 conferred disease risk in an autosomal recessive fashion: Odds ratio was 10.1 (95% CI 4.36, 23.06), adjusted for SNP CFH 402, for those carrying two copies of the risk allele, whereas indistinguishable from unity if carrying only one risk allele. Conclusions The HTRA1 promoter polymorphism, rs11200638, is a strong candidate with a functional consequence that predisposes Japanese to develop neovascular AMD.

State College, PA) or through direct sequencing using CEQ2000XL DNA analysis system (Beckman Coulter, Fullerton, CA).
Hardy Weinberg equilibrium (HWE) χ 2 values in the entire sample and controls only were calculated to identify possible genotyping errors. No extreme deviations (χ 2 >50) were observed (Table 1). Linkage disequilibrium (LD) was measured by the D' value. For each SNP, Pearson's χ 2 tests with one degree of freedom for association were performed. Odds ratios (OR), population attributable risks (PAR), and their respective confidence intervals were calculated, formula in [8].
Previous functional data lead us to focus further analyses on rs11200638 [6,7]. Joint ORs for two SNPs (rs11200638 and CFH 402, previously genotyped) were calculated using standard methods [9]. Marginal ORs and their confidence intervals for the two SNP were calculated using logistic regression with SNP CFH 402 and rs11200638 as independent variables [9]. PARs were calculated using standard methods [9]. Confidence intervals around the PARs were constructed using 999 bootstrap replicates. To control for confounding, the Mantel-Hanzel test for association with two variables was used [9]. Four genotypic models were considered (Full, Recessive, Multiplicative, and Dominant) and the Aikake information criterion (AIC) was utilized to assess the fit of each model. All R scripts used in the analysis are available upon request.

RESULTS
SNP rs11200638, approximately 6.1 kb downstream of the surrogate SNP rs10490924, resides in the promoter of the HTRA1 serine protease gene (512 base pairs upstream of transcriptional start site). These two SNPs were in almost complete linkage disequilibrium (LD) and showed strong association with AMD in the Hong Kong study [6] and in a Caucasian population from Utah [7].
In our cohort, the two SNPs were also in complete LD, from which only two major (frequency >5%) haplotypes, one predominant in cases and one in controls, were observed. Disease association tests yielded p-values of 4.74x10 -11 and 1.79x10 -12 for rs10490924 and rs11200638, respectively (Table  1). Given the previous evidence of higher conservation across species [6] and the functional consequence of rs11200638 on HTRA1 expression [6,7], additional analyses focused on this SNP.
Reanalyzing the original CFH genotype data, we found the OR covered unity ( Table 2) and all interval estimates of PAR for CFH 402 variants under the four genotypic models included zero (Table 3). Of the four models, the best fit to the HTRA1 SNP genotypic effects, as assessed by Akaike's information criterion, was the recessive model, from which the risk genotype was AA and non-risk was GG and GA (Table 3). Under the framework of recessive rs11200638 and the two observed genotypes for CFH 402, no interaction was detected between the two SNPs based on the likelihood ratio test (Table  3). Odds ratios for different genotypes of rs11200638 do not vary a great deal depending on the CFH 402 genotypes, and vice versa ( Table 2). In fact, the OR curves shown in Figure 1 indicate a "removable" interaction between the two SNPs, in which the original two OR curves become parallel (i.e. no     interaction after transformation with a logarithmic function). Overall, after adjusting for the CFH 402 SNP, individuals carrying the risk homozygote AA of rs11200638 are greater than 10 times more likely to have AMD than those with the other genotypes (Table 2).

DISCUSSION
These data reconfirm the association of the HTRA1 promoter SNP rs11200638, independent of the CFH 402 polymorphism, with wet AMD. The present study genotyped two previously identified disease associated SNPs in the chromosome 10q26 region. Both SNPs showed similar significance levels. The first SNP, rs10490924, resides in the hypothetical locus, LOC387715. Several studies have found significant association between AMD and this SNP [10][11][12]. So far only one transcript from this hypothetical locus has been identified in one experiment. No study has identified the transcript or protein in the retina, much less identified a change in the protein as a result of the SNP. Additionally, sequence comparisons of seventeen species presented in DeWan et al. show higher sequence ©2007 Molecular Vision Molecular Vision 2007; 13:545-48 <http://www.molvis.org/molvis/v13/a58/>  conservation surrounding rs11200638 compared to that around rs10490924 [6]. HTRA1 is expressed in the retina in humans [5] and mouse [6]. Computational analysis of the HTRA1 promoter indicate that this SNP resides in a CpG island and may result in a change in the binding site for transcription factors AP2 and SRF [6]. Preliminary functional data suggest that individuals homozygous for the risk-allele at rs11200638 exhibit increased expression of HTRA1 [6,7]. Therefore, given the existing functional data, it appears as if the HTRA1 promoter polymorphism, rs11200638, is likely the underlying functional polymorphism in the 10q26 region. However, the mechanism to neovascularization is yet to be understood and will require intense investigation to uncover its link to the wet form of AMD.